(1) Field of the Invention
This invention relates to the production, the harvesting and the purification of virus from virus-infected cell cultures, for example for experimental and therapeutic purposes, e.g. for the production of pharmaceutical formulations such as prophylactic or therapeutic inoculations. In particular aspects, the invention relates to methods and arrangements for the production of preparations of herpesviruses. Other aspects of the invention will be apparent from the description given below.
(2) Description of the Related Art Including Information Disclosed Under 37 CFR 1.97 and 37 CFR 1.98.
Several methods are known for producing live virus preparations all of which include extraction of the virus from a virus infected cell, e.g. Vero cells or MRC5 cells among others, for vaccine, therapeutic and other purposes.
U.S. Pat. No. 3,985,615 (Osaka Research Foundation: T Kubo et al) shows production of live attenuated varicella virus for vaccine use by culture comprising passage in guinea pig primary embryonic tissue cells.
U.S. Pat. No. 5,024,836 (Merck: W J McAleer et al) relates to production of lyophilized vaccine preparations based thereon. Also disclosed is the fact that aqueous solutions of live virus vaccines are known to be unstable during storage.
DD-209738 (Cent Cerc Bioprep: IV Patrascu) illustrates production of another type of herpesvirus, for use as vaccine against Marek's disease. The herpesvirus is produced by (a) culturing specific-pathogen-free chicken embryo cells on dextran microspheres; (b) inoculating the culture at 80% confluence with turkey herpes virus strain FC-126 (clone 1, IIIb); (c) collecting the infected cells in SPGA medium (sucrose, phosphate, glutamate, bovine albumin fraction V) when the cytopathic effect is 80%; (d) subjecting the suspension to three ultrasonic pulses of 1 minute duration at 2 minute intervals and centrifuging it to recover a first crop of vaccine; (e) resuspending the sediment in SPCA medium and repeating step (d) to obtain a second crop of vaccine (to increase the vaccine yield by almost 2%); (f) freezing the combined vaccines at −100° C. prior to determining the virus titer; and (g) diluting the SPCA medium and freeze drying.
JP06234659-A (Z H Handai Biseibutsubyo Kenkyukai) describes, in an example, production of herpesviral vaccine on human diploid fibroblast MRC-5 cells cultured in MEM medium at 37° C., comprising inoculation of varicella virus Oka strain seed virus at a MOI of 0.03 to MRC-5 cells and culture at 37° C. for 2 days. Virus is then suspended in a solution containing 6.4 g NaCl, 0.16 g KCl, 2.3 g Na2HPO4 12H2O, 0.16 g KH2PO4, 5 0.0 g sucrose, 1.0 g Na L-glutamate, 2.0 g gelatin, 25.0 g gelatin hydrolysate and 0.1 g EDA-3Na per L.
EP 0 573 107, U.S. Pat. No. 5,360,736 and U.S. Pat. No. 5,607,852 (Merck: P A Friedman et al) describe processes for production of attenuated varicella zoster virus vaccine, including a process for preparing live, attenuated, cell-free varicella-zoster virus (VZV) vaccine that comprises: (a) Culturing VZV infection-susceptible cells, selected from human diploid cells, to confluency in monolayer culture, under conditions of sufficiently high nutrition to achieve a high degree of cell replication, and supplying a non-metabolizable disaccharide; (b) infecting the cells cultured according to step (a) at as close to the point of confluency as possible with as high a multiplicity of infection of VZV-infected cells as practical; (c) maintaining the VZV-infected culture in a state of high nutrition for about 22-96 hours and harvesting at the point of peak infectious VZV production; (d) washing the VZV-infected culture with a physiologic solution, optionally containing a lysosomotropic agent, such as ammonium chloride or chloroquine, prior to harvesting the VZV infected cells; (e) Harvesting the VZV infected cells into a minimal volume of a stabilizing solution and either disrupting the cells immediately or freezing the cells for later disruption, (1) Disrupting the VZV-infected cells to optimally release cell-associated VZV, and removing cellular debris, to provide a cell-free VZV preparation. The process is proposed for mass production of live vaccine. Appropriate nutrient medium for growing cells in monolayer culture in that connection is described as consisting essentially of SRFE-2 medium supplemented with between 0.2 mg/mL and 0.4 mg/mL soybean lipid and 10% fetal calf serum, the cells being selected from MRC-5 cells, WI-38 cells and Vero cells.
U.S. Pat. No. 5,665,362 (Cantab Pharmaceuticals Research: S C Inglis et al) and U.S. Pat. No. 5,837,261 (Cantab Pharmaceuticals Research: SC Inglis et al) disclose recombinant cells and culture methods for producing genetically disabled herpesvirus such as herpes simplex virus for vaccine purposes, wherein the virus is grown on complimentary cells.
U.S. Pat. No. 6,267,967 (Cantab: M D Johnston et al) describe processes for purification of herpes simplex virus. Infectious preparations of human herpesviruses such as herpes simplex virus (HSV), e.g. HSV type 2 (HSV-2), which tend to remain strongly cell-associated when grown in culture and affinity reagent carrying the virus, which can be applied from a carrier liquid containing salt (e.g. sodium chloride or other pharmaceutically acceptable salt over about 0.4M) or containing heparin or another sulfated or sulfonated polysaccharide (e.g. in the order of about 10-250, such as about 50, micro-g/mL), can then suitably be washed and the virus recovered in actively infectious form by elution, e.g. with high-concentration salt solution or with sulfated or sulfonated polysaccharide. The initial harvesting of virus from such a cell culture can be carried out in any of a variety of ways. Examples of methods include cell rupture, e.g. by freeze-thaw cycles or osmotic stress procedures, e.g. with hypotonic saline or glycerol solutions, sonication, elution by heparin or dextran sulfate or equivalent, or by using elution with saline solution.
U.S. Pat. No. 6,013,265 (UMB: L Aurelian), incorporated herein by reference, or U.S. Pat. No. 6,054,131 (UMB: L Aurelian), incorporated herein by reference, disclose a growth compromised herpes simplex recombinant virus, e.g. HSV-2, in which the PK domain has been deleted. In a wild type HSV-2 virus, replication began at 2 hours post-infection and reached peak levels at 36 hrs post-infection. In the PK deleted HSV-2, the onset of replication was not seen until 15 hrs post-infection both in 10% serum and 0.5% serum supplemented Vero cells. When replication resumed this mutant reached titers similar to those of HSV-2 at 36 hrs post infection in the presence of 10% serum, but not in cells supplemented with in 0.5% serum in the medium.
It remains desirable to provide methods for production of herpesvirus-containing preparations, capable of contributing to the manufacture of infectious virus preparations in good yield and purity, e.g. those that are to be used in vaccines or as therapies.